GSM8082757: Day 5 iPSC IFNg_BS; Mus musculus; OTHER
DNA was extracted using Wizard® Genomic DNA Purification Kit (Promega, A1120). After measuring DNA quantity by Qubit (ThermoFisher Scientific), 2µg of each sample was evenly splitted for the oxidation reaction (Oxidative Bisulfite -OxBS- treated samples) and the mock-oxidation reaction (Bisulfite -BS- treated samples) where the oxidant solution was replaced by water following the TrueMethyl oxBS Module manufacturer's instructions (NuGEN-Tecan, 0414). Both aliquots were then processed in parallel for all stages of the protocol. After the oxidation reaction where 5-hydroxymethylcytosine is oxidized to 5-formylcytosine (5fC) and 5-methylcytosine (5mC) stays unchanged, the bisulfite treatment converts 5fC and all non-methylated cytosines to uracil, while 5mC is not altered. Paired oxidative Bisulfite (OxBS) and the mock-oxidation reaction (Bisulfite BS) treated DNA from day 5 (control and IFNγ) reprogramming samples were used as templates for a two-step PCR amplification with the IMMOLASE™ DNA Polymerase (BIO-21046) with a custom program adapted to each primer pair annealing temperature and resulting amplicon size. In the first PCR, 0.5 ng of DNA were used to amplify the regions of interest to increase the specificity of the second PCR reaction and enrich the region of interest. In the second PCR, 1-3 µl of the first-step products were amplified using staggered primers to provide sequence variability for the sequencing. The sets of staggered forward and reverse primers were designed to contain partial P5 and P7 adaptor sequences, respectively, at the 5' and 2-5 random nucleotides (N) before the gene-specific sequence. The amplicons from the different regions of interest were gel-purified, quantified, and pooled together for library preparation. 4 libraries corresponding to control and IFNγ samples (BS/BS-OX) at day 5 were prepared for sequencing, and subsequent steps were completed following the MiSeq System protocol (MiSeq Reagent Kit v3, Illumina). Next Generation Sequencing was performed using MiSeq (300bp, single-end).